Composition for inhibiting transmission of hepatitis B virus

ABSTRACT

The present invention relates to methods of inhibiting transmission of Hepatitis B virus using compositions comprising silver sulfadiazine and preferably further comprising a biguanide such as chlorhexidine and/or a detergent such as sodium deoxycholate. It is based, at least in part, on the discovery that silver sulfadiazine alone or in combination with chlorhexidine or sodium deoxycholate was shown to inhibit Hepatitis B virus DNA synthesis. The inhibitory effect of silver sulfadiazine was enhanced by combining silver sulfadiazine with either chlorhexidine or sodium deoxycholate.

DESCRIPTION

This application is a continuation-in-part of U.S. Patent applicationSer. No. 07/262,165 filed Oct. 18, 1988 now U.S. Pat. No. 4,952,411which is a continuation-in-part of U.S. Patent application Ser. No.07/018,624 filed Feb. 25, 1987 now abandoned.

BACKGROUND OF THE INVENTION

The present invention relates to compositions for inhibiting thetransmission of Acquired Immunodeficiency Syndrome (AIDS).

AIDS is a fatal catastrophic disease that presently infects millions ofpeople worldwide. Although initially concentrated in central Africa andin certain high risk groups in other geographic areas including theUnited States, AIDS is now spreading to other areas and is appearing inindividuals who are not members of the recognized risk groups. As aresult, major efforts are being made to develop methods of preventingthe transmission of AIDS, methods of curing AIDS once contracted, andmethods of ameliorating the symptoms of AIDS. To date, however, AIDS hasproven difficult to treat or prevent.

AIDS is caused by a virus. This virus has been referred to by a numberof names in the literature, including HIV (human immunodeficiencyvirus), LAV (lymphadenopathy-associated virus), ARV (AIDS-related virus)and HTLV-III (human T-cell leukemia virus-III). For simplicity, thevirus causing AIDS will be referred to herein as the AIDS virus.

It is generally known that viruses can be divided into two groups basedupon the nature of the virus' genetic material. Some viruses are DNAviruses, that is, their genetic material is deoxyribonucleic acid, whileothers are RNA (ribonucleic acid) viruses. The RNA viruses can furtherbe divided into two groups, those in which replication of the vitalgenome proceeds by making an RNA copy directly from the RNA genome andthose in which a DNA intermediate is involved. This latter type of RNAvirus is called a retrovirus.

The AIDS virus is a retrovirus. Thus, like other retroviruses, it has anenzyme called reverse transcriptase (or RNA-dependent DNA polymerase)which catalyzes transcription of viral RNA into double helical DNA. ThisDNA sequence is integrated into the genome of the infected cell where itis known as a provirus. Subsequent transcription of this provirus by thetranscription mechanism of the infected cell produces new vital RNA forpackaging into new virus particles.

Because the AIDS virus may lie dormant in an infected cell in the formof a provirus for extended periods of time, it has been difficult toestablish the precise routes by which AIDS is spread. It is known,however, that AIDS can be transmitted to a person by transfusing thatperson with blood containing the AIDS virus. AIDS can also betransmitted to a person through homosexual or heterosexual intercoursewith a partner infected with the AIDS virus. Transmission of the AIDSvirus is facilitated by preexisting sexually transmitted diseases(STD's) other than AIDS, for example gonorrhea. Further, scientistssuspect that the AIDS virus is spread easily during sexual intercoursedue to tearing of tissue which would abet entry of the AIDS virus intothe blood stream.

In response to the growing threat of AIDS transmission, the use ofcondoms during sexual intercourse has been suggested as a means ofpreventing transmission of the AIDS virus. Improper use of condoms, ortheir perforation during intercourse renders them only partiallyeffective. Accordingly, there is a pressing need for a better method ofinhibiting the transmission of the AIDS virus in humans during sexualintercourse and during surgical procedures on infected patients. It isan object of the present invention to provide such a method.

SUMMARY OF THE INVENTION

The present invention provides an inexpensive, easily available andconvenient composition for inhibiting the transmission of the AIDS virusin humans for example, as a result of sexual intercourse. The inventionrelies upon a dual mode of action of particular compounds andcombinations thereof which results in a rapid killing action withinminutes. These compounds are effective to reduce the infectivity of theAIDS virus and also to kill the causative organisms of many other STD'safter short exposure. The method of the invention is therefore useful toreduce the immediate risk of AIDS transmission. It also reduces futurerisk of AIDS transmission by eliminating STD causing organisms whichincrease the risk of AIDS. The present invention also provides formethods of inhibiting transmission of Hepatitis B virus (HBV).

Silver salts, such as silver sulfadiazine (AgSD), are among thecompounds found to be effective antiviral agents against retrovirusesincluding the AIDS virus. Such materials had previously been recognizedas antibacterial agents useful in treating burns in man and animal. C.L. Fox, Jr., U.S. Pat. No. 3,761,590. AgSD has also been shown to beeffective against certain viruses such as herpes simplex and herpeszoster and against the causative organisms of many STD's includingCandida albicans, Treponema pallidum and gonorrhea. U.S. Pat. No.4,415,565 of Wysor shows further antiviral activity of AgSD againstcertain RNA viruses, but none of these are retroviruses. Thus, whileAgSD is a well studied material, there was no basis to expect that itwould have activity against the AIDS retrovirus which has proven sodifficult to inhibit or destroy.

Biguanides, such as chlorhexidine, have also been found to be effectivewhen used at sufficiently high levels as inhibitors of the AIDS virus.

We have also found that combinations of these compounds with each otherand with other antibacterial agents lead to an unexpected enhancement ofthe antiviral activity of AgSD and also in a rapid killing action.Specifically, AgSD in combination with chlorhexidine, a broad spectrumantibacterial agent, is substantially more effective for reducing theinfectivity of the AIDS virus than AgSD alone, despite the fact thechlorhexidine alone has no effect on infectivity of AIDS virus under thesame conditions. Increased effectiveness was also noted for combinationsof AgSD with detergents such as deoxycholate.

In view of these findings, the invention contemplates inhibiting thetransmission of AIDS comprising topically applying an effectiveantiviral amount of biguanide or a silver salt such as silversulfadiazine, alone or in combination. Other agents such as deoxycholatemay also be used. The composition is advantageously administered to asexual canal of a human prior to or during sexual intercourse. Thisapplication can be carried out by introducing a cream or foam into thesexual canal, or by coating the inhibitory composition onto a condom orother device that is inserted into the sexual canal.

BRIEF DESCRIPTION OF THE FIGURE

FIG. 1 is a graph of the rate of incorporation of radiolabeled thymidineby hepatitis B virus following exposure of the virus to AgSD alone or incombination with other agents.

DETAILED DESCRIPTION OF THE INVENTION

As noted above, the composition of the present invention is effective toinhibit the transmission of AIDS virus in humans and other mammals whenapplied topically in an effective antiviral amount. The compositioncomprises a biguanide, alone or in combination with other activeingredients.

As used in this application, the term sexual canal refers to either avaginal or an anal canal.

The antiviral composition used in the method of the invention comprisesbiguanide, such as chlorhexidine or a salt thereof.

The composition may also include a silver salt. While the exampleshereinbelow use one specific silver salt, AgSD, other silver salts mayalso be used. Other suitable silver salts include silver acetate, silverbenzoate, silver carbonate, silver chloride, silver iodate, silveriodide, silver lactate, silver laurate, silver nitrate, silver oxide,silver palmirate, and silver salts of proteins.

The antiviral composition of the invention preferably also comprises oneor more additional ingredients which enhance the antiviral effectivenessof the silver salt. Thus, the antiviral composition may containdetergents such as deoxycholate or benzalkonium chloride. Suitable saltsof these materials may also be used.

The antiviral composition may also include other materials which areeffective against STD-causing organisms which will reduce the long termrisk of AIDS infection. Examples of such materials include nonoxynol,which is effective against gonococcus and quinolones which are effectiveagainst numerous STD-causing organisms. It should be noted thatchlorhexidine and the detergents noted above are also effective againsta variety of STD-causing organisms, including herpes simplex virus(HSV), Hepatitis B virus (HBV) and Candida albicans. As demonstratedinfra in Example 6, AgSD alone or in combination with chlorhexidine orsodium deoxycholate was shown to inhibit HBV DNA synthesis. As shown inFIG. 1, the inhibitory effect of AgED was enhanced by combining AgEDwith either chlorhexidine or sodium deoxycholate. Accordingly, thepresent invention provides for methods of inhibiting transmission of HBVusing compositions comprising AgSD and preferably further comprising abiguanide such as, for example, chlorhexidine and/or a detergent suchas, for example, sodium deoxycholate.

The antiviral compositions for use in the invention can be applied as(a) a dispersion in a water-dispersible hydrophilic carrier; (b) as adispersion in a substantially water insoluble carrier; (c) as adispersion in a semi-soft or cream-like water-dispersible orwater-soluble oil-in-water emulsion carrier; or (d) as a dispersion inan aqueous sucrose carrier, e.g. an approximately 25%-50% by weightaqueous sucrose solution. Specific examples of formulating silversulfadiazine in various carriers are provided in U.S. Pat. No. 3,761,590which is incorporated herein by reference. The carrier will preferablycontain from about 0.1 to about 10% by weight of the silver salt and upto 2% of other active agents.

The antiviral composition useful in the method of the invention can becontained in a squeezable tube having an applicator nozzle. Thisfacilitates topical application of the composition to the sexual canalprior to intercourse by inserting the nozzle into the sexual canal andsqueezing the tube to force the antiviral composition into the sexualcanal. Alternatively, the antiviral can be applied with any of variousknown applicators for delivering drugs into a sexual canal. Theantiviral composition can also be topically applied during sexualintercourse by coating the penis itself or coating a condom with alubricant material, such as K-Y Jelly (Johnson & Johnson), that containsthe silver salt.

The antiviral composition of the invention may also be introduced intothe sexual canal as a coating on a device intended for insertion in thesexual canal. Examples of such devices include condoms, medical gloves,and diaphragms. Such devices may be coated or impregnated with theantiviral composition by spraying the completed device or byincorporating the antiviral composition during manufacture. Specifictechniques for preparing the devices are described in U.S. patentapplication Ser. No. 154,920, filed Feb. 11, 1988, and itscontinuation-in-part filed Oct. 14, 1988, both of which are incorporatedherein by reference.

Coating compositions can be made based upon polymeric carrierscontaining polyurethane or silicons. For example, a coating compositionfor use on condoms in accordance with the invention can be prepared bycombining chlorhexidine acetate (CHA) and N-ethyl-2-pyrrolidone (NEP)and heating to dissolve the CHA. Tetrahydrofuran (THF) is then mixed tothe CHA solution in NEP and the mixture is thoroughly added to form auniform solution. A polyurethane such as Pellethane® 2363-80AE (DowChemical Co.) is dissolved with heat in THF and a silver sulfadiazinepowder may then be added to form a suspension. The CHA/NEP solution andthe polyurethane suspension are then combined to form the coatingcomposition.

Silicones can be used to provide a coating which is lubricious andreleases the drug in a controlled dosing manner. Mixtures of Silastic®Medical Adhesive Type A, a polydimethyl siloxane, and MDX-4-4159, afluid silicone comprising equal parts of an amino functionalpolydimethyl siloxane copolymer and a mixed aliphatic and isopropanolsolvent are suited as polymeric coating agents. A 1:1 mixture of thesesilicones provides a film with desirable biocompatible characteristics.

The experimental results which demonstrate the effectiveness of theclaimed method are set forth below. These tests involve the AIDS virus,a recognized model system for the AIDS virus or a recognized STDorganism. Further, although the tests with the AIDS virus itself arenecessarily in vitro tests in view of the catastrophic consequences ofAIDS, these in vitro tests are highly predictive of and correlate within vivo efficacy. They thus support the surprising finding thatcompositions containing biguanides with or without silver salts can beused to inhibit transmission of AIDS as a result of sexual intercourse.

EXAMPLE 1

The effectiveness of AgSD against the AIDS virus J1 in vitro wasassessed by testing the infectivity of samples of HTLV-III in H9 cellsafter exposure to AgSD for 10 minutes. Due to the relatively low titersachievable with the AIDS virus, it was necessary to devise means forseparating the bulk of the AgSD from the virus to be assayed. After anumber of preliminary experiments, it was found that the best method ofthose investigated was to rapidly pass the AgSD/AIDS virus mixture overa Sephadex G-25M column, recover the AIDS virus containing void volumeand precipitate the virus using polyethylene glycol (PEG).

To determine recovery of the virus using this method, a controlpreparation containing virus but no AgSD was similarly processed.

It was also necessary to confirm that this procedure was effective toremove all of the AgSD. This was accomplished using "Stop Controls".This involved processing AgSD alone through the column, precipitatingthe same fraction with PEG and then adding active AIDS virus to theprecipitate. If the titer of the stop control had been similar to thecontrol preparation containing virus but no AgSD it would have indicatedthat little or no AgSD was present in the precipitate. In fact, however,the titer was substantially lower in the stop controls (Samples 4 and 6)than in the corresponding test samples without silver sulfadiazine(Samples 1 and 2). This indicates that some of the silver sulfadiazineis not being separated. While this means that virus killing occurredover a longer period than the ten minute contact time, it also suggeststhat the virucidal activity is fairly strong to persist even at thereduced levels.

The specific tests conducted are summarized in Table 1. For each sampleto which virus was added initially, the virus sample was a stocksolution prepared from a 10,000 fold concentrate of HTLV-III obtainedfrom Bionetics Research. This material was diluted 1:10 with ConditionedInfection Medium (CIM) to form a stock solution with an actual virustiter of 10⁵.5 /ml. Two AgSD stock preparations were also prepared, a 1%by weight in 50% by weight aqueous sucrose preparation and an 0.5% byweight in 25% by weight aqueous sucrose preparation.

To conduct the tests, 60 μl aliquots of the virus stock were placed inmicrofuge tubes as samples 1-3 and 6 as indicated in Table 1. This wasmixed with 540 μl of the respective AgSD preparations in tubes 3 and 5and with 540 μl of CIM in tubes 1 and 2. Tubes 4 and 6 each received 600μl of the respective AgSD preparations, but no virus. Each tube was thenmixed with a vortex mixer and allowed to incubate for 10 minutes at roomtemperature.

To separate the AgSD from the virus, the contents of each tubecontaining AgSD then centrifuged in a microfuge for 1 minute, and thesupernatants were collected. These supernatants and the entire sample oftube 2 were then introduced onto a Sephadex-25M column. The columns usedhad a fitted disc at the top of the column and a void volume ofapproximately 1 ml. These columns are normally stored in sodium azideand had been prepared by washing under sterile conditions with 18successive 4 ml portions of CIM medium on the day prior to theexperiment.

Each of the samples was placed on the column until it passed through thefitted disc. The column was then eluted with 4 ml of CIM medium. Thefirst 3 ml of eluent was discarded and the last ml was collected into asterile microfuge tube containing 0.35 ml of 30% PEG 6000 in phosphatebuffer. These tubes were held at 0° C. for at least 30 minutes and thencentrifuged for 1 minute in a microfuge. The pellets were collected andresuspended in either 0.5 ml CIM (samples 2, 3 and 5) or in an HTLV-IIIcontaining medium made by diluting 0.7 parts of the virus stock with 6.3parts of CIM.

Each of the six samples thus prepared was assayed in quadruplicate with10-fold dilutions in CIM for its ability to infect H9 cells. This wasdone by adding 50 μl of a preparation containing 2.4×10⁶ /ml H9 cellsthat had been conditioned in CIM for 1 hour at 37° C. to each 100 μl ofsample or dilution. This culture was fed 25 μl of CIM on days 4, 7 and10. On day 4, cytotoxicity was evaluated by visual examination of thecultures.

The results of these observations are shown in Table 1. As can beclearly seen, AgSD substantially reduced the infectivity of AIDS virustested without any observation of cytotoxicity.

EXAMPLE 2

The effect of AgSD, chlorhexidine and sodium deoxycholate, bothindividually and in combination, on the infectivity of the ARV-2 strainof AIDS virus was tested in H9 cells using lower concentrations of drugsuch as can be practically coated onto a glove or condom or otherdevice. These concentrations were below the level that producedsubstantial observable cytotoxicity, even during incubation with thevirus, and yet were effective at killing the virus.

A stock solution of virus containing 3 to 5×10⁴ infectious virusparticles/ml was preincubated with the various drugs as indicated inTable 2 for 15 minutes. The virus sample was then diluted 4-fold inorder to reduce the concentrations of the drugs below levels toxic to H9cells (see Example 3 below) and mixed with 250,000 H9 cells in a totalvolume of 1 ml. After 24 hours, the cells were assayed to determine thepercentage of the culture expressing viral antigen. This time intervalwas selected as it allows for only a single round of viral infection tohave occurred such that the number of cells infected was a directreflection of the number of infectious virions present in the originalsample.

As can be seen from Table 2, AgSD alone at these low concentrations wasonly slightly effective, but better results were obtained when AgSD wasused in combination with either sodium deoxycholate and chlorhexidine.Of particular significance is the marked reduction in infectivityobserved for the combination of AgSD (5 μg/ml) and chlorhexidine (5μg/ml) since chlorhexidine (10 μg/ml) did not itself reduce viralinfectivity.

EXAMPLE 3

The toxicity of the various agents used in the antiviral compositions ofthe invention to human T₄ -lymphocytes (H9 cells and marophages whichare the carriers of the AIDS virus) may be relevant to the effectivenessof a drug. This is because killing these cells when present in semen orvaginal fluids may lead to release of virus making it more susceptibleto the effects of the drug. With this in mind, the effect of shortexposure (10 minutes) of AgSD and other drugs on H9 cells was tested bytreating a suspension of H9 cells (1.6×10⁶ /ml in HBSS) with 50 and 100ml/ml of each drug or drug combination. After incubating for 10 minutes,the cells were washed twice in thirty volumes of HBSS; resuspended inRPMI 10% FCS+NaPyruvate and plated into 24 well plates at 4×10⁵cells/mi. Cell viability was determined after 24 hours and is reportedas numbers of viable cells per ml and viable percentage (live cells/livecells+dead cells) in Table 3A. As can be seen, each of the agents testedkills some of the cells, although the most significant killing isobserved for 100 μl/ml AgSD and the combination of AgSD and sodiumdeoxycholate.

The effectiveness of killing of macrophages was also tested as shown inTable 3B. In the experiment, peritonial normal mouse macrophages wereenriched by attaching to petri dishes and adjusted to a cellconcentration of 5 to 10×10⁶ /ml. 0.1 ml aliquots of this suspensionwere plated in microtiter plates and 10μ and 5μ of each of four sampleswas added. The control plate received PBS only. After 20 minutes ofincubation in a CO incubator, the cells were tested for viability usingtryphan-blue dye. The percent kill is shown in Table 3B.

EXAMPLE 4

In vivo tests were performed using Rauscher Leukemia Virus (RLV), arecognized retrovirus model (see, e.g., Nature 323, 467-469 (1986);Rupecht et al., Proc. Nat'l. Acad. Sci. USA 82, 7733-7737 (1985)) whichis used by the FDA in testing drugs for use in treating AIDS. RLV wasintroduced into Balb/CICR mice in which it infects the spleen. The levelof virus infectivity was quantified by determining the weight increaseof the mouse spleen after 20 days from infection.

A preliminary experiment was first carried out to determine the effectof the drugs to be tested on the spleen. Nine sets of five mice each (6week old female mice) received 0.25 ml injections into the tail vein ofone of an extract of a glove treated with one of the followingsolutions:

1. Silver Sulfadiazine (2%)

2. Sodium Deoxycholate (2%)

3. Chlorhexidine (2%)

4. Silver Sulfadiazine (1%)+Sodium Deoxycholate (1%)

5. Silver Sulfadiazine (1%)+Chlorhexidine (1%)

6. Fusidic Acid (2%)

7. Fusidic Acid (1%)+Chlorhexidine (1%)

8. Saline incubated glove

9. Saline-no glove

Each treatment was prepared by incubating 1.5 ml Dulbecco's PhosphateBuffered Saline (PBS) for 10 minutes at 37° C. in the finger tip of alatex glove. After incubation, as much as possible of the material wasremoved from the glove. 0.4 ml of PBS was then introduced into the gloveand this was the sample which was introduced into the animals. Theanimals that did not receive a clean stick during the injection wereexcluded from the study. Thus two of the groups only had four animalseach that were considered.

Eight days after injection each of the animals was sacrificed and thespleen weights determined for each animal. No increase in spleen weightwas observed in any of the groups.

An additional eleven groups of 5 mice each were then used to test theeffectiveness of these same compounds against infectivity of RLV. Eachtreatment was prepared by incubating 0.4 ml sterile PBS containing RVB3(a strain of RLV) for 10 minutes in a glove tip which had previously hadone of drugs or straight PBS incubated in it as described above. Threeadditional controls, a PBS containing glove with no virus, a virussample not incubated in a glove, and a PBS sample not incubated in aglove were also run. The mice in this case were sacrificed 20 days afterinjection and spleen weights determined as shown in Table 4. Each of thematerials tested showed a substantial reduction in virus infectivity.

EXAMPLE 5

The combination of AgSD with chlorhexidine and deoxycholate was alsofound to be particularly effective against several STD-causingorganisms. As shown in Tables 5A and 5B silver sulfadiazine incombination with chlorhexidine or sodium deoxycholate is particularlyeffective against Candida albicans. Similarly, these combinations areeffective to kill Gonococcus (Table 6) and herpes virus (Tables 7A and7B).

EXAMPLE 6

The effect of AgSD alone or in combination with chlorhexidine or sodiumdeoxycholate on DNA synthesis by Hepatitis B Virus was studied bymeasuring the rate of incorporation of radiolabeled thymidine. As aresult, it was found that the AgSD interferes with the RNA-dependent DNApolymerase of Hepatitis B virus, an interference which is enhanced byusing it in combination with either chlorhexidine or sodium deoxycholate(FIG. 1).

EXAMPLE 7

The effect of chlorohexidine on HIV-I was tested by researchers atStuart Pharmaceuticals, Wilmington, Del. using a 4% chlorohexidinegluconate (CHG) hand scrub (HIBICLENS®) and an 0.5% CHG-containing handrinse (HIBISTAT®). In each case, an HIV-I preparation was exposed todilutions of one of the two materials for 10 minutes after which theviral preparation was used to infect C3-44 cells. The presence of HIV-Iinfection was monitored by indirect immunofluorescense by detectingvital p24 antigen expression and by reverse transciptase activity inculture fluid as a measure of virus production. The results of thisexperiment showed that, chlorohexidine gluconate at concentrations of0.04%, 0.05% and higher were effective to prevent HIV-I infection, whileconcentrations of 0.01% and lower were not. Thus, it appears that athreshold level of chlorohexidine is necessary for activity and that theresults in Example 2 can be attributed to the use of chlorohexidine at alevel below this threshold.

EXAMPLE 8

A silicone coating agent was prepared by dispersing 2.5 ml of Silistic®Medical Adhesive Type A in 55 ml of THF to which 2.5 ml of MDX-4-4159 isadded. 4 g of AgSD are suspended in 30 ml and 2 g of CHA are dissolvedin 10 ml of ethanol. The AgSD suspension is mixed with the siliconedispersions and finally the CHA solution is added dropwise while thepreparation is agitated. Either 5% NEP or 5% DMAC can be substituted forethanol in the above formulation.

The coating agent prepared above was used to apply a coating oncatheters fabricated from silicone, polyurethane and latex substrates.The coatings were applied by dipping and then drying. Results are givenin Table 8 below.

                  TABLE 8                                                         ______________________________________                                        Antibacterial Efficacy of Polyurethane                                        I.V. Catheters and Latex or Silicone Urinary                                  Catheters Coated with A silicone Matrix                                       Catheter Type                                                                              Drug in Catheter                                                                            Days of Activity*                                  ______________________________________                                        Polyurethane I.V.                                                                          CHA           2                                                  Polyurethane I.V.                                                                          AgSD + CHA    4                                                  Latex urinary                                                                              AgSD          2                                                  Latex urinary                                                                              AgSD + CHA    4                                                  Silicone urinary                                                                           AgSD          3                                                  Silicone urinary                                                                           AgSD + CHA    4                                                  ______________________________________                                         *Determined via Bioassay A. Inoculum used to assay urinary catheter is a      10.sup.4 CFU of a 1:1 mixture of Staph. epi and E. coli; 10.sup.4 CFU of      Staph. aureus is used to challenge the I.V. catheter.                    

EXAMPLE 9

The fingers of latex gloves were washed and dried. They were thensprayed with a fine mist spray of a coating solution to provide auniform coating of solution on the glove surface, sufficient to providecomplete wetting thereof without runoff. The coating solutions wereprepared by dissolving 1% Silicate®Medical Adhesive Type A and 1% of thesilicone MDX4-4159 in ethyl acetate, followed by dissolving anddispersing the chlorhexidine acetate and silver sulfadiazine,respectively, therein. The coating was air dried for 24 hours and thegloves tested using the following test:

Treated glove fingers were draped over the tops of culture tubes withthe treated side with sprayed on coating forming the inside of the cupshape. Then 3.0 ml of TSB containing 10⁴ colony forming units of Staph.aureus was dispensed in each finger and all placed in a water bathshaker at 37° C. Samples were removed at 15 minutes, 1 hour, 2 hours,and 4 hours, diluted 1-10, and the solution plated on blood agar in 2.0ml amounts.

The results of the test are summarized in the following Table 9.

                  TABLE 9                                                         ______________________________________                                        Antibacterial Efficacy of Drug Coated                                         Gloves against Staph. aureus                                                                 Colony Counts in Culture                                       Drug in Coating Solution                                                                       15 min. 1 hour  2 hours                                                                             4 hours                                ______________________________________                                        None (Control)   12,000  15,000  20,000                                                                              50,000                                 Chlorhexidine (1%)                                                                             100     0       0     0                                      Silver Sulfadiazine (2%)                                                                       3,300   200     0     0                                      Silver Sulfadiazine (1%) +                                                                     0       0       0     0                                      Chlorhexidine (1%)                                                            ______________________________________                                    

It is noted that the gloves coated according to this procedure wereflexible and met all other requirements for high quality latex gloves.

                                      TABLE 1                                     __________________________________________________________________________    ASSAY MIXTURES AND RESULTS                                                                HTLV-III                 PEG Pellet                                                                            Log.sub.10 **                    Sample      (Stock 21)                                                                          Mixture            Resuspended                                                                           TCID.sub.50                                                                         Log***                     No. Material                                                                              10-1  CIM   AgSD                                                                              Stop Procedure                                                                         in (0.5 ml)                                                                           Per/ml                                                                              Kill                                                                              Cytotoxicity           __________________________________________________________________________    1   HTVL-III                                                                              60 ul 540 ul                                                                              --  --       --      4.5   --  0                          (Stock 21)                                                                    (10.sup.-1                                                                2   HTVL-III                                                                              "     "     --  Column + Peg                                                                           CIM     4.25  --  0                          (Stock 21)                                                                    (10.sup.-1                                                                3   1% AgSD "     --    540 Cent.* + Peg                                                                           CIM     2.0   2.25                                                                              0                          in 50%                                                                        aqueous                                                                       sucrose                                                                       solution                                                                  4   1% AgSD --          600 "        10.sup.-2 HTLV-III                                                                    3.25  --  0                          in 50%                           (Stop Control)                               aqueous                                                                       sucrose                                                                       solution                                                                  5   0.5% AgSD                                                                             60 ul --    540 "        CIM     2.25  2.0 0                          in 25%                                                                        aqueous                                                                       solution                                                                  6   0.5% AgSD                                                                             --          600 "        10.sup.-2 HTLV-III                                                                    3.75  --  0                          in 25%                           (Stop Control)                               aqueous                                                                       solution                                                                  __________________________________________________________________________     *Centrifuge 1 minute in microfuge  place supernatant on column                **TCID.sub.50 = Tissue Culture Infecting Dose.sub.50                          ***Compared to Sample No. 2                                              

                  TABLE 2                                                         ______________________________________                                        Drug During   Final       %        % Infection                                Incubation                                                                             (μg/ml)                                                                             (Drug) μg/ml                                                                           Infection                                                                            v. Control                               ______________________________________                                        chlorhexidine                                                                          10       2.5         3.35   108                                      (CHA)                                                                         sodium   40       10.0        3.35   108                                      deoxycholate                                                                  (NaDC)                                                                        AgSD     10       2.5         2.95   95                                       AgSD +   10       2.5 +       2.85   92                                       NaDC     40       10.0                                                        AgSD +    5 +     1.25 +      2.45   72                                       CHA       5       1.25                                                        ______________________________________                                    

                  TABLE 3A                                                        ______________________________________                                                 Viable                     %                                                  Cells/ml                   Viab**                                    ______________________________________                                        *AgSD 50   4 × 10.sup.5                                                                    Cells in terrible condition.                                                                   37                                        100        5 × 10.sup.4                                                                    "                 0                                        CHA 50   1.5 × 10.sup.6                                                                    "                73                                        100      2.5 × 10.sup.5                                                                    "                20                                        NaDC 50  1.2 × 10.sup.6       73                                        100      2.0 × 10.sup.6       44                                        AgSD 50 +                                                                              1.5 × 10.sup.4        0                                        CHA 50                                                                        H.sub.2 O                                                                              3.1 × 10.sup.6       89                                        Cells Alone                                                                            3.0 × 10.sup.6       88                                        ______________________________________                                         *AgSD + insoluble. In an attempt to remove drug cells were spun at 200 g      for 15 sec. (including acceleration and decleration time) + Cell pipetted     off, then washed two times.                                                   **live cells/live & dead                                                 

                  TABLE 3B                                                        ______________________________________                                        Results                                                                       Rate of Killing of Macrophage by Drugs                                                            % Kill                                                    ______________________________________                                        Control                36                                                     AgSD (100 μg)      100                                                     CHA (100 μg)       100                                                     AgSD + CHA (50 μg + 50 μg)                                                                     85                                                     ______________________________________                                    

                                      TABLE 4                                     __________________________________________________________________________    Results                                                                                              Weight of                                                                             Weight                                                      Concentration of                                                                        Spleen (mg)                                                                           Increase                                                    Drug in Coating                                                                         (Average of                                                                           from                                           Drug in Glove                                                                              Solution (%)                                                                            6 Animals)                                                                            Control (mg)                                   __________________________________________________________________________    Silver sulfadiazine                                                                        2         106     20                                             Deoxycholate 2         109     23                                             Chlorhexidine                                                                              2         234     148                                            Silver sulfadiazine +                                                                      1 + 1     115     29                                             deoxycholate                                                                  Silver suladiazine +                                                                       1 + 1     103     17                                             chlorhexidine                                                                 Fusidic acid 2         107     21                                             Fusidic acid +                                                                             1 + 1     319     23                                             Chlorhexidine                                                                 Control glove +        86      0                                              PBS medium                                                                    No glove - only PBS    86      0                                              medium                                                                        Control glove + RVB3   1,627   1,541                                          No glove + RVB3        1,280   1,194                                          __________________________________________________________________________

                  TABLE 5A                                                        ______________________________________                                        Rate of Killing of Candid-albicans                                            by silver sulfadiazine an other                                               agents on short exposure                                                                    Concen-   Colony Counts in Culture                              Drug          tration   (10 Minute Incubation)                                ______________________________________                                        Silver sulfadiazine                                                                         100       10,000                                                Chlorhexidine 100       30                                                    Deoxycholate  1,000     8,000                                                 AgSD + Chlorhexidine                                                                        50 + 50   0                                                     AgSD + Deoxycholate                                                                         100 + 100 20                                                    Nonoxynol     0.2%      >50,000                                               Control                 >50,000                                               ______________________________________                                         3 ml of Saboraud broth containing 10.sup.5 organism of Candida albicans       were incubated with the above drug. Aliquots were removed at 5 and 10         minute and were subcultured.                                             

                                      TABLE 5B                                    __________________________________________________________________________    Antibacterial Efficacy of Drug Coated Gloves                                  against Candida albicans                                                      Treated glove fingers were draped over the top of culture                     tubes with the treated side foring the inside of the cup                      shape. Then 3.0 ml of TSB contining 10.sup.3 organisms of Candida             albicans was dispensed in each finger and all placed in the                   water bath shaker at 37° C. Samples were removed at 15                 minutes, 1 hour, 2 hours, and 4 hours. They were diluted 1-                   10 and plated on blood agar in 2.0 ml amounts.                                            Colony Counts in Culture                                          Drug in Glove                                                                             15 Minutes                                                                          1 Hour 2 Hours                                                                              4 Hours                                       __________________________________________________________________________    None (Control)                                                                            1,400 2,000  4,000  6,000                                         Chlorhexidine                                                                             75    0      0      0                                             Silver Sulfadiazine                                                                       1,650 1,500  1,500  2,200                                         Silver Sulfadiazine +                                                                     0     0      0      0                                             Chlorhexidine                                                                 Silver Suladiazine +                                                                      1,500 400    0      0                                             Deoxycholate                                                                  Silver Suladiazine +                                                                      0     0      0      0                                             Chlorhexidine +                                                               Nonoxynol                                                                     __________________________________________________________________________

                  TABLE 6                                                         ______________________________________                                        Killing of Gonococcus by                                                      Silver Sulfadiazine and Other Agents                                                           Colony Counts in Culture                                     Drugs      μg/ml    5 Minutes  10 Minutes                                  ______________________________________                                        AgSD       100         4,000      2,000                                       Deoxycholate                                                                             1,000       12,000     4,000                                       Chlorhexidine                                                                            100         2,000      10                                          Nonoxynol  0.1%        40         70                                          AgSD +     50 + 50     0          0                                           Chlorhexidine                                                                 AgSD +     100 + 1,000 10         0                                           Deoxycholate                                                                  None (Control)         >50,000    >50,000                                     ______________________________________                                         Drugs were suspended in 5 ml of cultures containing 105 organisms of          gonoccccus and incubated. Aliquots were removed at 5 and 10 minute            intervals and subcultured for colony counts.                             

                  TABLE 7A                                                        ______________________________________                                        Toxicity of Drugs for HSV                                                     One ml HSV at 3 × 106/ml was incubated with 200 μliters of           drugs each 500 μg/ml stock solution. After 20 minutes at                   R.T., the virus was titered on monolayers of vero cells,                      incubatwd for 2 hours, then overlayed with methyl cellulose.                  Virus titers were read after 48 hours. No drug toxicity* was                  seen in rows titer read in.                                                   μliters added to 1 ml Virus                                                                  Titer    % Inhibition                                       ______________________________________                                        200 AgSD          5.2 × 10.sup.5                                                                   81                                                 200 Chlorhexidine 2.7 × 10.sup.6                                                                   0                                                  100 AgSD +        1.5 × 10.sup.4                                                                   99.5                                               100 Chlorhexidine                                                             200 NaDC          3.2 × 10.sup.6                                                                   0                                                  100 NaDC + 100 AgSD                                                                             1.3 × 10.sup.6                                                                   54                                                 100 NaDC +          8 × 10.sup.4                                                                   93                                                 100 Chlorhexidine                                                             200 Benzalkonium chloride                                                                       5.2 × 10.sup.4                                                                   98                                                 200 H.sub.2 O     2.8 × 10.sup.6                                                                   0                                                  200 Media         3.3 × 10.sup.6                                                                   0                                                  ______________________________________                                         *Drug conc. in firt row was 4-8 μg/ml                                 

                  TABLE 7B                                                        ______________________________________                                        Effect of HSV-1 of Interaction with Drug Treated Gloves                       HSV-1 w diluted to 3 × 10.sup.6 PFU/ml in DME 10% FCS.                  On ml of virus was placed in sterile drug treated gloves,                     incubated for 10 min. at room temperature then titred                         on Vero cells.                                                                Treatment        Titer (PFU/ml)                                               ______________________________________                                        virus (no glove) 2.9 × 10.sup.6                                         virus + ccntrol tube                                                                           3.0 × 10.sup.6                                         virus + tube w   4.3 × 10.sup.6                                         virus + tube x   <10                                                          virus + tube y   <10                                                          ______________________________________                                         W = Silver sulfadiazine                                                       X = Silver sulfadiazine + Deoxycholate                                        Y = Silver sulfadiazine + Chlorhexidine                                  

It is claimed:
 1. A method for inhibiting transmission of hepatitis Bvirus, comprising applying to a patient a composition comprising aneffective antiviral amount of silver sulfadiazine at a site wherecontact with hepatitis B virus may occur.
 2. A method according to claim1, in which the composition further comprises a biguanide in an amountsuch that the combination of silver sulfadiazine and biguanide has anantiviral effect.
 3. The method according to claim 2, in which thebiguanide is chlorhexidine.
 4. The method according to claim 1, in whichthe composition further comprises chlorhexidine in an amount such thatthe combination of silver sulfadiazine and chlorhexidine has anantiviral effect.
 5. The method according to claim 1, in which thecomposition further comprises a detergent selected from the groupconsisting of sodium deoxycholate and benzalkonium chloride.
 6. Themethod according to claim 2, in which the composition further comprisesa detergent selected from the group consisting of sodium deoxycholateand benzalkonium chloride.
 7. The method according to claim 6, in whichthe composition further comprises a detergent selected from the groupconsisting of sodium deoxycholate and benzalkonium chloride.
 8. Themethod according to claim 4, in which the composition further comprisesa detergent selected from the group consisting of sodium deoxycholateand benzalkonium chloride.
 9. The method according to claim 1, in whichthe composition further comprises chlorhexidine and sodium deoxycholatein an amount such that the combination of silver sulfadiazine,chlorhexidine and sodium deoxycholate has an antiviral effect.